The general principles of what was originally termed "antisense" therapy are now well recognized. Most diseases and undesirable conditions in humans and animal subjects are mediated by specific DNA or RNA sequences which, if inactivated, would no longer be able to facilitate the progress of the disease. The "antisense" approach provides DNA or RNA oligomers, or their analogs, which are capable of specific binding to the undesirable nucleic acid sequences; recently the possibility of specific binding of oligomers to protein targets has also been explored. These materials can be supplied directly or generated in situ, and may be conventional oligomers, or are more commonly oligomers having properties which make them, for example, resistant to nucleases or more capable of specific binding to the desired target. The specific binding may be effected by providing oligomers having sequences which result in conventional base-pairing, or these may recognize double-stranded DNA by binding to the major or minor grooves which are present in the double helix, or the oligomers, in either single stranded or duplex form may recognize target protein. Whatever the ultimate strategy, it is desirable to provide oligomers with physiological properties which render them more effective. Hence, the concept has expanded beyond a simple "antisense" approach to include any therapy by oligonucleotides. The general approach to constructing various oligomers useful in antisense therapy has been reviewed by vander Krol, A. R., et al., Biotechniques (1988) 6:958-976, and by Stein, C. A., et al., Cancer Res (1988) 48:2659-2668, both incorporated herein by reference.
The art provides a number of approaches whereby modified oligonucleotides are used in these expanded antisense applications. For example, in order to provide enhanced stability in vivo, through resistance to endogenous nucleases, oligomers have been synthesized with alternative linkages other than the conventional phosphodiester linkage. Among these are the methylphosphonates wherein one of the phosphorous-linked oxygens has been replaced by methyl; phosphorothioates, wherein sulfur replaces one of the oxygens; and various amidates, wherein NH.sub.2 or organic amine derivatives, such as morpholidates or piperazidates, replace an oxygen. Also carbonate and carbamate linkages have been employed, as well as those involving sulfur rather than oxygen as a linking substituent.
In addition, modifications have been employed wherein the oligonucleotides are conjugated with a lipophilic group to enhance cell permeation capability. Inclusion of intercalators and chelators which enhance the ability of the oligonucleotide to bind the target DNA or RNA is also known. These substituents have been attached to the 5' end of preconstructed oligonucleotides using amidite or H-phosphonate chemistry, as described by Ogilvie, K. K., et al., Pure and Appl Chem (1987) 59:325, and by Froehler, B. C., Nucleic Acids Res (1986) 14:5399. Intercalators have also been attached to the 3' end of oligomers, as described by Asseline, U., et al., Tet Lett (1989) 30:2521. This last method utilizes 2,2'-dithioethanol attached to a solid support to displace diisopropylamine from a 3' phosphonate bearing the acridine moiety and is subsequently deleted after oxidation of the phosphorus. Other substituents have been bound to the 3' end of oligomers by alternate methods, including polylysine (Bayard, B., et al., Biochemistry (1986) 25:3730; Lemaitre, M., et al., Nucleosides and Nucleotides (1987) 6:311) and, in addition, disulfides have been used to attach various groups to the 3' terminus, as described by Zuckerman, R., et al., Nucleic Acids Res (1987) 15:5305. It is known that oligonucleotides which are substituted at the 3' end show increased stability and increased resistance to degradation by exonucieases (Lancelot, G., et al., Biochemistry (1985) 24:2521; Asseline, U., et al., Proc Natl Acad Sci USA (1984) 81:3297).
Recently, two papers have suggested the use of amino protected derivatives of 3-amino-l,2-propanediol for the insertion of an amino group capable of further derivatization to desirable labeling or other moieties. Nelson, P.S., et al., Nucleic Acids Res (1989) 17:7187-7194, describe the conjugation of the diol to a modified form of controlled pore glass, linked through the intermediation of succinic anhydride, wherein the resulting controlled pore glass is used as a synthesis support to obtain an oligonucleotide containing, ultimately, a reactive amino group at the 3' terminus. This technique permits double labeling of the oligonucleotide at both 3' and 5' ends wherein the 5' terminus is labeled by other means. In another report, Nelson, P.S., et al., Nucleic Acids Res (1989) 17:7179-7186, describe the incorporation of the protected cyanoethoxydiisopropyl aminophosphenyl derivative of 3-amino-l,2-propanediol into the nucleotide chain. This provides a free amino group at any location for subsequent derivatization. In this report, the amino-modified oligonucleotide was subsequently labeled with biotin. These constructions result in a chiral center and create a mixture of diastereomers.
The phosphodiester linkages of native DNA and RNA molecules are readily degraded by exonucleases present in cells, tissue culture media, serum, blood and other body fluids. For example, exonuclease activity in tissue culture media containing serum results in extensive DNA or RNA oligomer degradation within about 30 minutes to 6 hours. Synthetic oligodeoxynucleotides with conventional phosphodiester linkages can be readily used in genetic engineering, for example, to locate specific RNA or DNA fragments from a library, since these oligonucleotides are usually not exposed to the relatively stringent environment of the culture medium; however, therapeutic uses in humans or animals and research applications in tissue culture require nucleic acid molecules that are stable under these conditions for more than several hours or days.
For example, oligonucleotides can be used to block protein synthesis by hydrogen bonding to complementary messenger RNA (mRNA) thereby providing a therapeutic agent for use in an antisense mode. Exonuclease-stable oligonucleotides could also be utilized to form triple-helix nucleic acid complexes which would interfere with the transcription process or with DNA replication by competing with naturally occurring binding factors or polymerases, or by irreversible gene inactivation. In order to utilize synthetic oligonucleotides in this manner, they must be stable to intracellular and extracellular exonucleases.
The major exonuclease activity associated with cells and body fluids appears to progress 3' to 5' on the substrate oligonucleotide. Although exonucleases which progress from the 5' to 3' termini of the substrate oligonucleotide are detectable in cell culture media under tissue culture conditions, this type of degradation appears to play a smaller role in vivo in whole organisms, as described in the parent application, Ser. No. 07/482,943.
Much research has been devoted to the synthesis of oligonucleotides that are nuclease-stable. This work has centered on modification of internucleotide phosphodiester linkages in order to render the linkage resistant to enzyme-mediated hydrolytic attack. As expected, modified internucleotide linkages are generally nuclease resistant. For example, Miller et al., Biochemistry (1981) 20:1874-1880, describe the synthesis and properties of oligodeoxyribonucleoside methylphosphonates, which oligomers were shown to be resistant to nuclease activity in vivo. Letsinger et al., Nucleic Acids Res (1986) 14:3487-3499, describe hydrogen bonding to complementary sequences and nuclease stability properties of short oligomers (dimers and trimers) possessing pendant groups linked to the oligomer at internucleotide phosphodiester linkages. These modified phosphodiester linkages were shown to be resistant to individual endo- and exonucleases compared to unmodified linkages under in vitro conditions. Stein et al., Nucleic Acids Res (1988) 16:3209-3221, describe the nuclease resistance properties of oligodeoxynucleotides modified to contain phosphorothioate internucleotide linkages. Although the phosphorothioate compounds were stable in vitro to individual nucleases, the binding affinity to complementary nucleic acid sequences was significantly reduced. Agrawal et al., Tet Lett (1987) 28:3539-3542, show enhanced endonuclease and exonuclease stability for oligodeoxynucleotides containing methyl-phosphonate-modified internucleotide linkages under in vitro conditions.
Walder et al., PCT publication WO89/5358, describe oligonucleotides modified at the 3' terminal internucleotide linkage that are more stable than control compounds with unmodified phosphodiester linkages. Copending application U.S. Ser. No. 07/361,045, assigned to the same assignee, describes oligonucleotides containing phosphoramidate linkages at the 3' terminal linkages which are stable to nucleases under both intracellular and extracellular conditions. Matteucci, Tet Lett, (1990) 37:2385-2388, describes the synthesis and properties of oligodeoxynucleotides containing formacetal internucleotide linkages which are exonuclease stable under in vitro conditions.
In the foregoing reports, nuclease stability results from modification of internucleotide linkages. In other instances, nuclease stability was obtained through alternate modifications. Copending application U.S. Ser. No. 07/425,857, by Buhr and Matteucci, describes by modification of the 2' hydroxyl positions of the oligomers. Haralambidis et al., Tet Lett (1987) 28:5199-5202, describe the attachment of a peptide to the 3' hydroxyl terminus of an oligonucleotide which was resistant to a 3' exonuclease (snake venom phosphodiesterase) under in vitro conditions. Further analysis of these oligonucleotide-peptide conjugates by Haralambidis et al., Nucleic Acids Res (1990) 18:493-499, showed they were, however, sensitive to P1 3' exonuclease. In none of these studies was stability of the compounds to serum or intracellular conditions (the conditions pertinent to stability for .in vivo therapeutic use) tested, although it is known generally that modifications at the 3' terminus offer enhanced stability (supra).
The present invention provides modified oligonucleotides which bear useful substituents at the 3' or 5' end, or at any intermediate position, by virtue of inclusion of at least one pseudonucleoside unit in the construction of the polymer, which, in turn provides a means for conjugation of desirable substituents such as intercalators, lipophilic groups, or chelators, in addition the invention provides additional 3' modified oligomers with enhanced nuclease resistance.